长期深低温冷冻对外周血造血干与祖细胞采集物中细胞活性及淋巴细胞亚群的影响

The influences of long-term cryopreservation on cell viability and lymphocyte phenotypes of peripheral blood HS/PC collections.

目的探讨长期深低温冷冻保存对动员后外周血造血干/祖细胞采集物中有核细胞活性、粒、巨噬细胞-集落形成单位(CFU-GM)生成能力及淋巴细胞免疫表型的影响。方法新鲜采集的标本作为对照组(12例),实验组按冷冻保存时间分为两组:实验1组(保存3～5年,26例)、实验2组(保存5～7年,9例)。冷冻标本经解冻、新鲜标本经裂解红细胞后流式细胞术(FCM)测病死率(7AAD+)和凋亡率(caspase-3+),CFU-GM 培养,流式细胞术检测淋巴细胞免疫表型:CD3/CD4/CD8/CD19/CD16+56,CD4/CD45RA/CD45RO,CD8/CD28。结果对照组有核细胞(NC)坏病死率和凋亡率显著低于两实验组(P<0.05),实验2组凋亡率(35.60±17.51)%高于实验1组(24.14±16.87)%(P=0.031)。CFU-GM/105NC在这三组分别为:139.25±122.7、117.26±182.08、29.51±48.65。CD34+细胞的比例对 CFU-GM/105NC有显著相关性(对照组 r=0.76,P=0.029;实验1组 r=0.849,P=0.000)。三组中淋巴细胞免疫表型分析差异无统计学意义。结论长期冷冻保存会影响有核细胞的活性和造血干与祖细胞的功能,但仍可保持一定的淋巴细胞免疫表型。

Objective To study the influences of long-term cryopreservation on nucleated cell viability,colony-form- ing units-granulocyte-macrophage(CFU-GM)-forming capability and Lymphocyte immune-phenotypes of pe- ripheral blood hematopoietic stem/progenitor cell(HS/PC)collections.Methods Fresh samples(n=12)were as control,frozen samples were divided into two groups:group 1(cryopreserved for 3-5 years,n=26)and group 2 (cryopreserved for 5-7 years,n=9).Flow cytometry(FCM)method based on 7 AAD or caspase-3staining was ap- plied to assess the degree of cell necrosis or apoptosis in cryopreserved and red-cell lytic collection samples.Lympho- cyte immune-phenotypes(CD3/CD4/CD8/CD19/CD16+56,CD4/CIM5RA/CIMSRO,CD8/CD28)were also de- tected by FCM.CFU-GM culture was done to study the hematopoietic activity.Results Percentages of necrosis and apoptosis cells of the control were both less than the groupl and group2(P<0.05); apoptosis cell of group2 was more than group 1[(35.60±17.51)% to(24.14±16.87)% P=0.031].The results of CFU-GM/103NC were 139.25± 122.7(control)、117.26±182.08(group1)、29.51～48.65(group2)and percentage of CD34+cells correlated well with CFU-GM/105NC in the control and groupl.There was no statistically significant difference in immunopheno- types among three groups.Conclusion Long-term cryopreservation may influence nucleated cell viability and hemato- poietic activity,but may still retain some lymphocyte phenotypes.